In the article I, we tested our first atherosclerotic mouse model, apoE-Leiden. The model was moderate and only early complicated lesions were found. Still, in the light of various postulated functions of apoE, the presence of endogenous mouse apoE synthesized in the liver, macrophages and some other tissues makes apoE3-Leiden mice a useful model for atherogenesis, because these animals offer a possibility to study the effects of increased lipid levels without disturbing the macrophage functions and LDL receptors. Furthermore, similarities in the pathogenesis with the human disease suggest that apoE3-Leiden transgenic mice are an attractive model for genetic, pathophysiological, and intervention studies of cardiovascular diseases.
Articles II-IV are focused on safety studies related to gene therapy in general and to VEGF-A in particular.
In the article II, the new method, in situ PCR, was tested. Today, gene transfer has been used for the treatment of several diseases. However, several safety concerns have been raised especially with regard to the stable integrated vectors. In situ PCR is a technique which combines the high sensitivity of PCR with the advantages of in situ hybridization, i.e. exact localization of the positive signal. In this study, we tested if this method can be used to localize integrated viral vector DNA theoretically even a year after the gene transfer. It was concluded that in situ PCR is a sensitive method to localize integrated viral vector DNA in both paraffin and frozen sections of the different tissues. Further, this precise histological localization of the signal is the most important quality of the method allowing comparison between transgene
6. SUMMARY AND CONCLUSIONS
In the article I, we tested our first atherosclerotic mouse model, apoE-Leiden. The model was moderate and only early complicated lesions were found. Still, in the light of various postulated functions of apoE, the presence of endogenous mouse apoE synthesized in the liver, macrophages and some other tissues makes apoE3-Leiden mice a useful model for atherogenesis, because these animals offer a possibility to study the effects of increased lipid levels without disturbing the macrophage functions and LDL receptors. Furthermore, similarities in the pathogenesis with the human disease suggest that apoE3-Leiden transgenic mice are an attractive model for genetic, pathophysiological, and intervention studies of cardiovascular diseases.
Articles II-IV are focused on safety studies related to gene therapy in general and to VEGF-A in particular.
In the article II, the new method, in situ PCR, was tested. Today, gene transfer has been used for the treatment of several diseases. However, several safety concerns have been raised especially with regard to the stable integrated vectors. In situ PCR is a technique which combines the high sensitivity of PCR with the advantages of in situ hybridization, i.e. exact localization of the positive signal. In this study, we tested if this method can be used to localize integrated viral vector DNA theoretically even a year after the gene transfer. It was concluded that in situ PCR is a sensitive method to localize integrated viral vector DNA in both paraffin and frozen sections of the different tissues. Further, this precise histological localization of the signal is the most important quality of the method allowing comparison between transgene
In the article III, the short-term possible atherosgenic effect of VEGF family members were studied in mouse model which most closely resembled the human profiles and shared the same arterial defendence. The role of vascular endothelial growth factors (VEGFs) in large arteries has been proposed to be either vasculoprotective or proatherogenic. Because the VEGFs are used for human therapy, it is important to know whether they might enhance atherogenesis. In conclusion, no evidence of increased atherogenesis was found in LDLR/apoB48-deficient mice after adenovirus-mediated systemic gene transfers of VEGF-A, -B, -C, and -D. The results are in line with findings from recent phase II/III clinical trials and animal studies.
Even though most human trials have used local intracoronary or intramyocardial delivery of VEGF genes or recombinant proteins, systemic delivery is the only feasible nontraumatic approach for gene or recombinant protein delivery in mice.
However, we recognize that our results do not exclude the possibility of VEGF-derived harmful effects potentially caused by higher doses or alternative delivery routes. It is obvious that the possibility of enhanced atherogenesis should be carefully monitored in forthcoming clinical trials involving VEGF recombinant proteins or genes. The present results do not suggest any increased proatherogenic effects of the members of the VEGF gene family when expressed transiently after systemic adenoviral gene transfer.
In the article IV, a new mouse model was developed in order to study the potential long-term side effects of therapeutic hVEGF-A165gene transfer. The VEGF family has been shown to play a major role in vascular permeability, angiogenesis, and lymphangiogenesis both during embryonic development and in adults. VEGFs have also been used in clinical applications as recombinant proteins or gene therapy. In conclusion, we have created a transgenic mouse model which over-expresses hVEGF-A165after Cre-protein gene transfer. In this model, the levels of hVEGF-A165
expression were moderate and most mice were healthy without any major consequences except typical angiogenic changes in the liver up to 18 months.
In the article III, the short-term possible atherosgenic effect of VEGF family members were studied in mouse model which most closely resembled the human profiles and shared the same arterial defendence. The role of vascular endothelial growth factors (VEGFs) in large arteries has been proposed to be either vasculoprotective or proatherogenic. Because the VEGFs are used for human therapy, it is important to know whether they might enhance atherogenesis. In conclusion, no evidence of increased atherogenesis was found in LDLR/apoB48-deficient mice after adenovirus-mediated systemic gene transfers of VEGF-A, -B, -C, and -D. The results are in line with findings from recent phase II/III clinical trials and animal studies.
Even though most human trials have used local intracoronary or intramyocardial delivery of VEGF genes or recombinant proteins, systemic delivery is the only feasible nontraumatic approach for gene or recombinant protein delivery in mice.
However, we recognize that our results do not exclude the possibility of VEGF-derived harmful effects potentially caused by higher doses or alternative delivery routes. It is obvious that the possibility of enhanced atherogenesis should be carefully monitored in forthcoming clinical trials involving VEGF recombinant proteins or genes. The present results do not suggest any increased proatherogenic effects of the members of the VEGF gene family when expressed transiently after systemic adenoviral gene transfer.
In the article IV, a new mouse model was developed in order to study the potential long-term side effects of therapeutic hVEGF-A165gene transfer. The VEGF family has been shown to play a major role in vascular permeability, angiogenesis, and lymphangiogenesis both during embryonic development and in adults. VEGFs have also been used in clinical applications as recombinant proteins or gene therapy. In conclusion, we have created a transgenic mouse model which over-expresses hVEGF-A165after Cre-protein gene transfer. In this model, the levels of hVEGF-A165
expression were moderate and most mice were healthy without any major consequences except typical angiogenic changes in the liver up to 18 months.
However, one mouse died spontaneously because of bleeding into abdominal cavity and having liver haemangioma, haemorrhagic paratubarian cystic lesions, and spleen peliosis. More importantly, two mice developed malignant tumors (hepatocellular carcinoma and lung adenocarcinoma), which were not seen in controls. Thus, we concluded that uncontrolled long-term expression of hVEGF-A165 may cause significant pathological changes in target tissues, and tight regulation of the transgene expression seems to be a prerequisite for all therapeutic applications aiming at long-term expression of hVEGF-A165.
However, one mouse died spontaneously because of bleeding into abdominal cavity and having liver haemangioma, haemorrhagic paratubarian cystic lesions, and spleen peliosis. More importantly, two mice developed malignant tumors (hepatocellular carcinoma and lung adenocarcinoma), which were not seen in controls. Thus, we concluded that uncontrolled long-term expression of hVEGF-A165 may cause significant pathological changes in target tissues, and tight regulation of the transgene expression seems to be a prerequisite for all therapeutic applications aiming at long-term expression of hVEGF-A165.
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